Blood samples are routinely taken in evacuated tubes, such as glass VACUTAINER.RTM. Brand evacuated sample collection tubes (Becton Dickinson and Company). One end of a double-ended needle is inserted into a patient's vein. The other end of the needle then punctures a septum covering the open end of the evacuated sample collection tube so that the vacuum in the tube draws the blood sample through the needle into the tube. Using this technique a plurality of samples can be taken using a single needle puncture of the skin. Plastic tubes have also been proposed for blood collection. Plastic offers several advantages related to lower breakage than glass tubes, less weight in shipment, and easier disposal by incineration for examples. Similarly, sample collection tubes designed for gravity based collection of smaller samples, such as a blood sample from an infant are also used with the same techniques. One such type of smaller tube is the MICROTAINER.RTM. Brand sample collection tube sold by Becton Dickinson and Company.
Blood samples collected in evacuated tubes are prepared for clinical examination in either the coagulated or non-coagulated (anticoagulated) state. In the latter category, which is the object of this invention, anticoagulation agents are utilized. There are a variety of anticoagulants routinely used in the art. These can be broadly classified as either calcium chelator or thrombin inhibitors. Calcium chelators function by removing calcium from solution in blood as a free ion. This inhibits coagulation because calcium is a necessary divalent ion for blood enzyme function. Citric acid, ethylenediaminetetraacetate (EDTA), fluorides, and oxalates are chemical anticoagulants that fall into calcium chelator category.
Thrombin is a serine protease derived from blood that converts fibrinogen to fibrin. Fibrin, in turn, polymerizes into a network that causes blood to gel or coagulate. As such, thrombin is a pivotal enzyme in hemostasis, and interruption of thrombin function can thus prevent blood coagulation. Thrombin inhibitors directly interfere with thrombin enzymatic function by blocking interaction with fibrinogen. Hirudin and heparin (through the antithrombin-heparin complex ATIII) are biochemical anticoagulants falling into the thrombin inhibitor category. Hirudin and heparin are well known in the field of hematology.
Calcium chelator and thrombin inhibitor anticoagulants have characteristic interferences with downstream clinical analysis. As examples, EDTA is successfully used in about 35% of hematology tubes (tripotassium, disodium, dipotassium, and liquid forms), but can induce blood cell aggregation. EDTA is not generally acceptable for use in nucleic acid diagnostics applying DNA amplification reactions such as polymerase chain reaction (PCR), which is important in growing markets of cellular analysis and viral load testing. Also, EDTA is inhibitory in T-cell stimulation assays. Oxalates can cause blood cells to shrink or expand, thus complicating cell volume measurements. Fluorides used in glucose testing are relatively poor anticoagulants and can cause hemolysis. Heparin can not be used in coagulation tubes because heparin interferes with enzymes of the coagulation cascade as well as with PCR.
Citrate is in widespread use for coagulation assays and is the anticoagulant of choice for PCR, but citrate causes some problems in cell or virus culture. Hirudin, a strong anticoagulant derived from leeches, and synthetic hirudin analogs are not yet in widespread commercial applications due to cost and availability.
Also, a nucleic acid ligands or aptamers to thrombin have been developed which inhibit coagulation of blood (Kubik, M. F. et al., Nuc. Acids Res. 22, 2619 (1994) and Bock L. C. et al., Nature 355, 564 (1992)). These nucleic acid ligands have not been used in any commercial products.
Another issue in anticoagulation is that of reversibility. In certain blood diagnostic tests it is useful to potentiate the coagulation cascade by reversing the anticoagulated state. For example, in the testing for blood coagulation dysfunctionality, it is customary in the art to use citrated blood plasma because the anticoagulated state can be rapidly reversed by addition of exogenous calcium which replenishes the necessary calcium for blood enzyme function. Anticoagulation of heparinized blood can be reversed by addition of heparinase, an enzyme that digests heparin and eliminates the coagulation-inhibiting heparin-ATIII complex. Unlike calcium and heparin, however, no reversing agents for hirudin are known. Thus hirudin is only appropriate for permanently anticoagulated blood specimens. Reversibility of the effects of nucleic acid ligands has also not been demonstrated in the art.
Blood testing would be significantly simplified if a single specimen of blood could be used for whole blood, plasma, and serum applications. Thus, there is need in the art for a "universal anticoagulant" that does not interfere with downstream clinical or hematological testing and is reversible by addition of a suitable reversing agent.